Any discussion of the prior art throughout the specification should in no way be considered an admission that such prior art is widely known or forms part of common general knowledge in the field.
The introduction of exogenous material such as small organic molecules, proteins and nucleic acids into cells in vitro and in vivo is crucial for the progression of research and development of therapies, as well as therapeutic delivery strategies.
For example, the introduction of fluorescently tagged proteins into cells allows real time analysis of the trafficking of the proteins throughout the cells, which may also assist in the identification of protein interactions during clinically important stages of a disease, or in response to specific triggers. Introducing putative small organic molecule drugs, which do not naturally cross cell membranes effectively, into cells during drug development can be informative on the activity of said drugs prior to diverting valuable time and effort towards developing delivery vehicles for these drugs.
The introduction of nucleic acids into cells is a critical step in cell therapy manufacturing, where expression vectors encoding genes are delivered across the cell membrane into the cytoplasm to effectively engineer live cells that can be used as therapeutic agents. By way of example, cell therapy may be used to induce an individual's own immune system to attack cancer cells or evade a virus, such as HIV. Cancer and HIV are of particular relevance from a global health perspective given their prevalence in the population with an estimated 35 million HIV patients in 2013 and 14 million new cancer cases in 2012. Utilising cell-derived gene therapy as part of a global health strategy requires a cell therapy manufacturing method capable of reproducibly producing sufficient quantities of product to potentially treat tens of millions of patients per annum at the appropriate price point and under current Good Manufacturing Practice in accordance with regulatory standards.
Accordingly, the ability to introduce exogenous material and in particular nucleic acids into cells in a quick and efficient manner is both a valuable research tool and a useful component of a therapeutic strategy.
There are several known methods for introducing agents into cells, with the choice of method generally being determined by the type of cell, the level of efficiency required, the size of the molecule being introduced and the number of cells available.
Although the terms may be used interchangeably, the introduction of agents such as nucleic acids into eukaryotic cells is generally referred to as “transfection”, whereas the introduction of nucleic acid into prokaryotic cells is generally referred to as “transformation”. Transfection and transformation methods may be conveniently separated into three categories, namely, chemical, physical and viral-based methods.
Chemical methods of transfection employ reagents such as cationic lipids, calcium phosphate, cationic polymers and dendrimer molecules to essentially package the nucleic acids for delivery into the cell. However, many of these methods are not applicable to all cell types. Moreover, they can be compromised by pH fluctuations or salts/phosphates in the cell media. Due to the requirement for packaging of nucleic acids in some of these methods, the size of the nucleic acid molecules that can be accommodated may be limited. Further, chemical transfection methods can require use of reagents that are expensive and/or toxic to cells in high concentrations and/or the method may only achieve low/inconsistent transfection efficiencies.
Conventional physical methods used to transfect eukaryotic cells include the use of magnetic nanoparticles, electroporation, bolistic particle delivery and microinjection. However, these methods tend to be quite harsh on the cells, often resulting in high mortality rates. These methods may also require immobilised cells, expensive equipment and/or a greater degree of technical skill on the part of the person performing the method. For example, in some electroporation methods, suspended cells are first permeabilised, followed by the application of an electric field to facilitate active delivery of charged exogenous material. Hence these techniques require specialised equipment and consumables for permeabilising the membranes of the cells and applying the electrical field.
Viral-based transfection methods rely on viral vectors including lentiviral, adenoviral and retroviral vectors for the delivery of nucleic acids into a cell, where the nucleic acids may be expressed at high levels by virtue of a viral promoter. These viral-based methods are expected to prove useful for the effective treatment of cancers of the lymphatic and haematopoietic systems, and for HIV therapeutics. However, due to variable transfection efficiencies, the cost of manufacturing viral vectors for these types of therapeutics is in the order of thousands of dollars per patient. Further, this method can be both labour intensive and prone to manufacturing issues if the process is not automated.
The introduction of exogenous material, and in particular, nucleic acids, into prokaryotic cells is also an important aspect for the manufacture of biologics during therapeutic drug development and indeed, research in general. Transformation of bacterial cell lines with exogenous nucleic acids for the recombinant production of valuable molecules such as biologic-based pharmaceuticals (so called biopharmaceuticals) can be achieved by various methods including chemical transformation and electroporation. However, these methods may require that the cells be made “competent” prior to transformation (e.g., by inducing high cell density and/or nutritional limitation which switches on a set of genes), they may not be applicable to all cell types and/or they may result in high levels of cell mortality.
Consequently, there is a need for a fast and efficient method of introducing exogenous material into a range of cell types that overcomes one or more of the difficulties of the known methods. Preferably, the method would deliver an acceptable level of cell viability and it would be cost-effective.
It is an object of the invention to overcome or ameliorate at least one of the disadvantages of the prior art, or to provide a useful alternative.
It will be appreciated that reference herein to “preferred” or “preferably” is intended as exemplary only.